Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance in Castration-Resistant Prostate Cancer

Abstract

Importance: The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics.

Objective: To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment.

Design, setting, and participants: Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes.

Exposure: Enzalutamide, 160 mg, daily orally.

Main outcomes and measures: Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels).

Results: The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations.

Conclusions and relevance: Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.

Figures

Figure 1

AR mutations detected in the cfDNA of mCRPC patients treated with enzalutamide. a) The most common aberrations detected by aCGH and deep AR gene sequencing of cfDNA from patients at baseline. b) Landscape of AR gene mutations detected in the cfDNA of mCRPC patients at baseline and at progression on enzalutamide. Schematic of the AR protein domains mapped onto the exonic structure, showing the pile-up of recurrent mutations detected in the ligand binding domain. Each colored circle represents a single mutation detected in a single sample (i.e. one patient may be represented several times if they harbor multiple mutations or the same mutation in multiple temporal samples). c) Box plot showing the detected allelic frequency of each recurrent mutation baseline and at progression, demonstrating that the majority of patients with AR mutations have at least 5% tumor-derived cfDNA (known as ctDNA). d) High correlation in AR mutation frequency between patient cfDNA technical replicates. Note that the three instances where the validation experiments did not redetect a previously identified mutation, it had a very low original frequency. In those examples, failure of validation likely reflects the low sampling probability of a heavily diluted or rare mutant allele.

Figure 2

Association of AR gene status at baseline with progression-free survival on enzalutamide. Swimmers plot showing selected copy number changes and AR mutation status in each patient at enzalutamide baseline, as determined by aCGH and deep AR sequencing respectively. *bona fide AR amplification call (log2ratio > 1.2) only possible in certain patients.

Figure 3

Clinically-informative genomic aberration detectable in the cfDNA of mCRPC patients at progression on enzalutamide. a) Integrative landscape of alterations from aCGH, AR gene sequencing, and targeted sequencing. Columns represent patient samples at progression on enzalutamide, ordered in brevity of progression-free survival on treatment. *no baseline sample available for VC-093. b) Cluster of activating mutations falling within the phosphorylation domain of CTNNB1. Bar plot below indicates that this is a pan-cancer hotspot region for mutations. †two mutations detected in the same patient (VC-017). c) Germline defects in DNA damage repair genes detected across 30 patients at progression on enzalutamide. Protein schematic shows locations of BRCA2 mutations. Bar plot below demonstrates evidence of somatic loss-of-heterozygosity in patient cfDNA at progression.